The significance of understanding biomarkers to take full advantage of them in disease diagnosis and treatment as well as in biomedical research is well recognized by the science community. CD38, originally identified as a differentiation marker for hematopoietic cells, is present in many other types of cells, and its deregulation is found to contribute to several different human diseases, including leukemia, social behavior defects, diabetes, and osteoporosis. Understanding the roles CD38 plays in various normal and pathological conditions has the potential to offer new ways to treat these diseases. So far, some knowledge about CD38 has been acquired, though limited, thanks to the efforts from researchers in the field. CD38 acts both as an enzyme and a receptor. The enzymatic activity can convert nicotinamide adenine dinucleotide (NAD) to adenosine diphosphate ribose (ADPR) and cyclic ADPR (cADPR), and NAD phosphate (NADP) to nicotinic acid adenine dinucleotide phosphate (NAADP). Both cADPR and NAADP are potent Ca2+ messengers that can trigger Ca2+ release from internal stores. As a receptor, when activated by certain ligands, CD38 can trigger the phosphorylation of intracellular proteins, including c-Cbl and mitogen-activated protein kinases (MAPK). However, the exact roles CD38 plays in most normal and pathological conditions are poorly understood at present, despite the accumulation of the knowledge mentioned above. Several unanswered questions prevent a clear and complete understanding of CD38 function. For example, how CD38 gains access to its NAD or NADP substrate, whether CD38 is present in intracellular organelles, and how the receptor function affects the enzymatic function. In this proposal, we will develop and use chemical tools, particularly NAD analogs that can covalently label CD38 in live cells, to address these important questions concerning CD38 biology. These CD38 probes have unique features, such as cell permeability, compatibility with ligand binding to CD38, and the ability to inhibit CD38 enzymatic activity. These small molecule probes can be used to track CD38 trafficking in real time in live cells, determine the intracellular distribution of CD38, and isolate and identify unknown CD38 ligands. These experiments cannot be easily achieved using other methods. Our studies will lead to a more detailed mechanistic picture of CD38 biochemistry, which will help to understand the function of CD38 in various normal and pathological conditions and potentially lead to new ways to treat diseases involving CD38, such as leukemia, diabetes, autism, and osteoporosis. In addition, the NAD metabolizing capability of CD38 can have a significant impact on other NAD-dependent enzymes, particularly the NAD-dependent deacetylases (sirtuins) and poly(ADP-ribose) polymerases (PARPs). Understanding how CD38 works should shed lights on the potential interactions between CD38 and other NAD-dependent enzymes.